C. Ian Ragan

Photolabelling of a mitochondrially encoded subunit of NADH dehydrogenase with [3H]dihydrorotenone

Mitochondrial NADH dehydrogenase from bovine heart was photolabelled with the inhibitor [3H]dihydrorotenone. A constituent of the hydrophobic domain of the enzyme of M r 33 000 was the major site of labelling. The identity of this protein with the mitochondrially encoded ND‐1 gene product was established by immunoblotting and immunoprecipitation with an antiserum raised to the expected C‐terminal sequence of the human ND‐1 gene product.

Structure of NADH-ubiquinone reductase (complex I)

Publisher Summary This chapter provides an overview of structure of nicotinamide adenine dinucleotide hydride (NADH)-ubiquinone reductase (complex I). It presents evidence that all NADH-ubiquinone reductases exhibiting high H+e - translocation stoichiometry will prove to be similar both in structure and mechanism, whatever their source. NADH binds in a hydrophobic pocket on the large subunit of the flavoprotein (FP) fragment, which is closely linked with the largest subunit of the iron–sulfur protein (IP) fragment. Electron transfer occurs to the flavin mononucleotid (FMN) and iron–sulfur clusters of the FP fragment and then to the transmembranous iron–sulfur proteins of the IP domain, which form the major exposed regions of the protein. The physiological reaction, namely, oxidation of NADH by long sidechain ubiquinones, can be conveniently assayed in membranes or in the isolated enzyme by replacing the natural acceptor with short-chain homologs or the alkyl analogs. Immunological assays of this enzyme content suggest that only 3% of mitochondrial protein is attributable to complex I and, therefore, that the isolated enzyme is to some extent inactivated or modified.

Molecular basis of mitochondrial myopathies: polypeptide analysis in complex-I deficiency.

Clinical and biochemical data are reported for three patients with mitochondrial myopathy. One patient presented only with exercise-induced muscle weakness, whereas the other two showed signs of multisystem disease. Polarographic determination of oxygen uptake in skeletal muscle mitochondria suggested complex-I (nicotinamide adenine dinucleotide [reduced] ubiquinone oxidoreductase) deficiency. Sodium dodecyl sulphate polyacrylamide gel electrophoresis and immunoblotting with antibody to the holoenzyme of complex-I and specific antibodies to certain of the Fe-S subunits of complex-I showed a relatively normal profile in the least affected patient and a generalised reduction in the intensities of all crossreacting bands in the other two patients. The most severely affected patient also showed a disproportionate and pronounced reduction in the 24 K Fe-S subunit. Clinical severity of muscle involvement correlated with the biochemical deficiency as determined polarographically and with the immunoblot appearances.

[53] Proteins, polypeptides, prosthetic groups, and enzymic properties of complexes I, II, III, IV, and V of the mitochondrial oxidative phosphorylation system

Publisher Summary Tabulation of proteins, polypeptides, prosthetic groups, and enzymic properties of complexes I to V is complicated because of uncertainties regarding purity and unaltered enzymic properties of these complexes as well as methods of analysis. Often, alternate procedures for isolation of a complex from several laboratories on the composition and properties of the same preparation of a complex are not available. Ideally, an enzyme complex is a relatively stable structural unit with a fixed composition and ratio of components and with an overall enzymic function for the expression of which all the component parts of the complex are needed. In practice, however, this is an extremely difficult definition to apply even to a multimeric and apparently pure enzyme because in most instances resolution to “subunits” is irreversible. The scarcity of information on the above complexes from independent laboratories using independent methods makes even the simplest assignments fraught with uncertainties. It discusses that functionally, complexes I to V should be expected to display the enzymic, regulatory, and inhibitor-response properties of comparable segments of an inner membrane preparation, such as phosphorylating submitochondrial particles. The chapter presents the comparison of regulatory functions between the isolated complexes and their counterpart segments in mitochondria or submitochondrial panicles that is difficult because sufficient and precise information is not available on the possible regulatory functions of the components of the respiratory chain, ion translocation systems, and the ATP synthesizing machinery. Vesicularized preparations of complexes I, III, and IV are considered to exhibit respiratory control releasable by uncouplers.

The Enzymes and the Enzyme Complexes of the Mitochondrial Oxidative Phosphorylation System

This chapter is concerned with a general discussion of the enzymes which catalyze oxidative phosphorylation in bovine heart mitochondria. These enzymes are located in the mitochondrial inner membrane and appear to exist as components of five enzyme complexes. Complexes I, II, III, and IV are segments of the electrontransport system. Complex V is essentially devoid of respiratory-chain electron carriers, and appears to be concerned with energy conservation and transfer and ATP synthesis. Several of the proteins associated with these enzyme complexes have been obtained in highly purified form. They are succinate dehydrogenase (Davis and Hatefi, 1971a; Hanstein et al., 1971b; Hatefi and Stiggall, 1976), NADH dehydrogenase (Hatefi and Stiggall, 1976; Hatefi and Stempel, 1969), cytochromes c 1 (Yu et al., 1972) and c, an iron-sulfur protein which is associated with complex III (Rieske, 1965), ATPase (Knowles and Penefsky, 1972; Senior and Brooks, 1970; Catterell and Pedersen, 1971; Tzagoloff and Meager, 1971), and a low-molecular-weight protein which is necessary for oligomycin sensitivity of membrane-bound ATPase (MacLennan and Tzagoloff, 1968). These preparations are water soluble and are discussed extensively in other parts of this book and elsewhere (Hatefi and Stiggall, 1976; Singer and Gutman, 1971; Singer et al., 1973a; Pedersen, 1975; Penefsky, 1974). In this chapter, they will be considered mainly in connection with their role as components of the above complexes.

The phospholipid annulus of mitochondrial NADH—ubiquinone reductase A dual phospholipid requirement for enzyme activity

A phospholipid requirement for the activity of several membrane-bound enzymes has been clearly established, e.g. [1,2] but the evidence for a phospholipid dependence of mitochondrial NADH dehydrogenase is conflicting at present. Reversible loss of NADH-ubiquinone-1 oxidoreductase activity following removal of phospholipids by phospholipase A treatment [3,4] or cholate and ammonium sulphate extraction [5] has been reported, but a water-soluble, lipid-free NADH dehydrogenase catalyzing ubiquinone reduction has been isolated by Baugh and King [6]. An approach to the study of lipid-protein interactions which avoids the potential problem of irreversible denaturation on removal of lipids is the lipid-substitution technique of Warren et al. [7]. We have applied this method to purified NADH-ubiquinone oxidoreductase (Complex I) [8] which is isolated as a lipoprotein complex. We are interested in investigating the role of lipids in mitochondrial electron transport for reasons other than the conflicting views on NADH dehydrogenase. Firstly, the respiratory chain complexes contain relatively high levels of cardiolipin whose specialized function (if any) is unknown. Secondly, the means by which respiratory complexes interact in the membrane is unclear. For example although ubiquinone-lO behaves as a kinetically homogeneous pool mediating hydrogen transfer from (separate) Complex I to Complex III [9], this picture is difficult to reconcile with the formation of a 1 : 1 binary complex from

A spin label study of the lipid boundary layer of mitochondrial NADH-ubiquinone oxidoreductase

Mitochondrial NADH-ubiquinone oxidoreductase (Complex I) is a lipoprotein enzyme containing phosphatidylcholine (PC), phosphatidylethanolamine (PE) and cardiolipin. Enzyme preparations containing endogenous cardiolipin and a range of either soyabean PC or dimyristoylphosphatidylcholine (DMPC) concentrations have been made. Using a spin-labelled fatty acid, two probe environments differing in mobility have been shown to be present. The fatty acid probe has a relative binding constant (or partition coefficient between lipid and protein) of unity. The boundary layer or lipid annulus reported by the probe has a value of approx. 300 lipid molecules per molecule of enzyme FMN in preparations containing soyabean PC, or DMPC above the phase transition temperature of the latter. In soyabean PC-replaced enzyme the apparent size of the boundary layer is independent of temperature between 30 degrees C and 14 degrees C but shows a modest increase to about 400 lipid molecules per molecule of FMN between 14 degrees C and 2 degrees C. Complex I replaced with high concentrations of DMPC gives non-linear Arrhenius plots of NADH-ubiquinone oxidoreductase activity. The results of the ESR experiments show that both boundary layer and bulk lipid must be motionally restricted for this to occur. Thus, the change in activity is probably not caused by an effect exerted directly on the catalytic activity of the enzyme but is more likely due to restriction of free diffusion of ubiquinone to its site of reduction.

Immunological assays of the NADH dehydrogenase content of bovine heart mitochondria and submitochondrial particles.

The NADH dehydrogenase content of mitochondria and submitochondrial particles is more difficult to measure than the content of other electron-transport complexes. The absence of unambiguously identifiable chromophores has, until recently, necessitated the use of more indirect measurements based on turnover numbers. For example, a turnover number of 8 X 10’. mm-‘. mol FMN-’ in the NADH-KsFe(CN), oxidoreductase assay was determined [l] for the soluble type 1 NADH dehydrogenase. From this value and the specific activity of NADH-KaFe(CN), oxidoreductase in submitochondrial particles, a value for the NADH dehydrogenase content of the latter was calculated (0.03 nmol/mg protein). That this value (as nmol dehydrogenase FMN/mg protein) was considerably less than the total acidextractable FMN content of these particles showed that the use of total FMN content was an unreliable way of estimating NADH dehydrogenase content. However, the turnover numbers of purified NADH dehydrogenase preparations, all containing similar concentrations of FMN, vary considerably with the purification procedure between 4 X 10’ for complex I and 1.5 X lo6 for the preparation of [2]. In the absence of clear evidence that the lower range represents inactivation, dehydrogenase contents based on NADH-KsFe(CN)6 oxidoreductase activities should be treated with reservation. NADH-ubiquinone oxidoreductase activity is probably also unreliable in this respect since the kinetics of this reaction are very dependent on the phospholipid environment [3]. Here we describe two assays of NADH dehydrogenase content using antibodies to purified complex I. From these values we calculate the true turnover

[34] Isolation of the iron—sulfur-containing polypeptides of NADH: Oxidoreductase ubiquinone

Publisher Summary This chapter describes the methodology employed for the stepwise fragmentation of Complex I and isolation of subunits containing FeS clusters. Complex I is structurally composed of three distinct fragments, a water-soluble FeS-flavoprotein (FP), a water-soluble FeS-protein (IP), and a water-insoluble fraction containing phospholipids and hydrophobic polypeptides (HP). The resolution of Complex I into FP, IP, and HP is achieved in the presence of chaotropes that are also used for further fragmentation of FP and IP. Chaotropes destabilize membranes and increase the water solubility of hydrophobic proteins by lowering the transfer entropy of hydrophobic structures from a lipophilic surrounding into the destructured water of the aqueous phase. Improvements in the resolution of IP are required, as none of the resolved proteins is completely pure. The procedures are optimized for pH, the detergents used, and the concentrations of detergent, Na trichloroacetate (NaTCA), urea, and ammonium sulfate.

Photolabelling of mitochondrial NADH dehydrogenase with arylazidophosphatidylcholine

Received 16 February 1981 1. Introduction The proton-translocating NADH dehydrogenase of bovine heart mitochondria has been isolated in sub- stantially unmodified form as complex I or the NADH-ubiquinone oxidoreductase complex [ 11. The isolated enzyme is a lipoprotein consisting of at least 26 different polypeptides [2] which bind a molecule of FMN and at least 5 iron-sulphur centres [3], 4 of which have been purified or partially purified [4]. Studies on the organisation of the constituent poly peptides both in the isolated enzyme and in the membrane have been carried out using hydrophilic probes such as diazobenzenesulphonate (DABS) [5] and the hydrophobic probe, iodonaphthylazide (INA) [6]. Objections to the use of INA have been raised [ 71 because of the rather polar nature and long life- time of the photogenerated nitrene. Thus, it is pos- sible that INA preferentially labels proteins at the membrane surface rather than those embedded in the hydrophobic region of the membrane although this may, in fact, not occur [8]. A potentially better membrane probe is a chemically reactive phospho- lipid analogue. Here, we have used a phosphatidyl- choline with 12-amino-N-(2-nitro-4-azidophenyl) dodecanoic acid in the 2 position [9] to photolabel isolated complex I. 2. Materials and methods Complex I was prepared as in [lo]. Lipid-deple- tion and lipid-supplementation of the enzyme were as in [l l] and [6], respectively. [3H]Arylazidophospha- tidylcholine (AAPC) was synthesised as in [ 121 with a specific radioactivity of 7.5 Ci/mmol. An ethanolic solution of AAPC was added to complex I(10 mg/ml 0.67 M sucrose/50 mM Tris-HCl (pH 8.0)) to a final